Growth regulatable recombinant bcg immunogenic compositions

ABSTRACT

Immunogenic compositions comprising growth regulatable recombinant intracellular pathogens that have been transformed to express recombinant immunogenic antigens of the same or other intracellular pathogens are provided. Exemplary immunogenic compositions include, but are not limited to, growth regulatable and growth limited recombinant intracellular pathogen immunogenic compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 60/744,552 filed Apr. 10, 2006, theentire contents of which are incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant Nos. Al31338and Al068413 awarded by the Department of Health and Human Services. TheGovernment has certain rights in this invention.

FIELD OF THE INVENTION

The present invention generally relates to immunogenic compositionsderived from recombinant intracellular pathogenic bacteria. Moreover,the immunogenic compositions of the present invention also comprisegrowth regulatable recombinant attenuated Mycobacteria includingauxotrophic, prototrophic and metabolically impaired strains. Theimmunogenic compositions of the present invention are useful in inducingimmune responses in hosts.

BACKGROUND OF THE INVENTION

It has long been recognized that parasitic microorganisms possess theability to infect animals thereby causing disease and often death.Pathogenic agents have been a leading cause of death throughout historyand continue to inflict immense suffering. Though the last hundred yearshave seen dramatic advances in the prevention and treatment of manyinfectious diseases, complicated host-parasite interactions still limitthe universal effectiveness of therapeutic measures. Difficulties incountering the sophisticated invasive mechanisms displayed by manypathogenic organisms are evidenced by the resurgence of various diseasessuch as tuberculosis, as well as the appearance of numerous drugresistant strains of bacteria and viruses.

Among those pathogenic agents of major epidemiological concern,intracellular bacteria have proven to be particularly intractable in theface of therapeutic or prophylactic measures. Intracellular bacteria,including the genus Mycobacterium, complete all or part of theirlifecycle within the cells of the infected host organism rather thanextracellularly. Around the world, intracellular bacteria areresponsible for untold suffering and millions of deaths each year.Tuberculosis is the leading cause of death from a single disease agentworldwide, with 8 million new cases and 2 million deaths annually. Inaddition, intracellular bacteria are responsible for millions of casesof leprosy. Other debilitating diseases transmitted by intracellularagents include cutaneous and visceral leishmaniasis, Americantrypanosomiasis (Chagas disease), listeriosis, toxoplasmosis,histoplasmosis, trachoma, psittacosis, Q-fever, and legionellosis.

Currently it is believed that approximately one-third of the world'spopulation is infected by Mycobacterium tuberculosis resulting inmillions of cases of pulmonary tuberculosis annually. More specifically,human pulmonary tuberculosis primarily caused by M. tuberculosis is amajor cause of death in developing countries. Mycobacterium tuberculosisis capable of surviving inside macrophages and monocytes, and thereforemay produce a chronic intracellular infection. Mycobacteriumtuberculosis is relatively successful in evading the normal defenses ofthe host organism by concealing itself within the cells primarilyresponsible for the detection of foreign elements and subsequentactivation of the immune system. Moreover, many of the front-linechemotherapeutic agents used to treat tuberculosis have relatively lowactivity against intracellular organisms as compared to extracellularforms. These same pathogenic characteristics have heretofore limited theeffectiveness of immunotherapeutic agents or immunogenic compositionsagainst tubercular infections.

Recently, tuberculosis resistance to one or more drugs was reported in36 of the 50 United States. In New York City, one-third of all casestested was resistant to one or more major drugs. Though non-resistanttuberculosis can be cured with a long course of antibiotics, the outlookregarding drug resistant strains is bleak. Patients infected withstrains resistant to two or more major antibiotics have a fatality rateof around 50%. Accordingly, safe and effective immunogenic compositionsagainst multi-drug resistant strains of M. tuberculosis are sorelyneeded.

Initial infections of M. tuberculosis almost always occur through theinhalation of aerosolized particles as the pathogen can remain viablefor weeks or months in moist or dry sputum. Although the primary site ofthe infection is in the lungs, the organism can also cause infection ofnearly any organ including, but not limited to, the bones, spleen,kidney, meninges and skin. Depending on the virulence of the particularstrain and the resistance of the host, the infection and correspondingdamage to the tissue may be minor or extensive. In the case of humans,the initial infection is controlled in the majority of individualsexposed to virulent strains of the bacteria. The development of acquiredimmunity following the initial challenge reduces bacterial proliferationthereby allowing lesions to heal and leaving the subject largelyasymptomatic.

When M. tuberculosis is not controlled by the infected subject it oftenresults in the extensive degradation of lung tissue. In susceptibleindividuals lesions are usually formed in the lung as the tuberclebacilli reproduce within alveolar or pulmonary macrophages. As theorganisms multiply, they may spread through the lymphatic system todistal lymph nodes and through the blood stream to the lung apices, bonemarrow, kidney and meninges surrounding the brain. Primarily as theresult of cell-mediated hypersensitivity responses, characteristicgranulomatous lesions or tubercles are produced in proportion to theseverity of the infection. These lesions consist of epithelioid cellsbordered by monocytes, lymphocytes and fibroblasts. In most instances alesion or tubercle eventually becomes necrotic and undergoes caseation(conversion of affected tissues into a soft cheesy substance).

While M. tuberculosis is a significant pathogen, other species of thegenus Mycobacterium also cause disease in animals including man and areclearly within the scope of the present invention. For example, M. bovisis closely related to M. tuberculosis and is responsible for tubercularinfections in domestic animals such as cattle, pigs, sheep, horses, dogsand cats. Further, M. bovis may infect humans via the intestinal tract,typically from the ingestion of raw milk. The localized intestinalinfection eventually spreads to the respiratory tract and is followedshortly by the classic symptoms of tuberculosis. Another importantpathogenic species of the genus Mycobacterium is M. leprae that causesmillions of cases of the ancient disease leprosy. Other species of thisgenus which cause disease in animals and man include M. kansasii, M.avium intracellulare, M. fortuitum, M. marinum, M. chelonei, and M.scrofulaceum. The pathogenic mycobacterial species frequently exhibit ahigh degree of homology in their respective DNA and correspondingprotein sequences and some species, such as M. tuberculosis and M.bovis, are highly related.

Attempts to eradicate tuberculosis using immunogenic compositions wasinitiated in 1921 after Calmette and Guérin successfully attenuated avirulent strain of M. bovis at the Institut Pasteur in Lille, France.This attenuated M. bovis became known as the Bacille Calmette Guérin, orBCG for short. Nearly eighty years later, immunogenic compositionsderived from BCG remain the only prophylactic therapy for tuberculosiscurrently in use. In fact, all BCG immunogenic compositions availabletoday are derived from the original strain of M. bovis developed byCalmette and Guérin at the Institut Pasteur.

The World Health Organization considers the BCG immunogenic compositionsan essential factor in reducing tuberculosis worldwide, especially indeveloping nations. In theory, the BCG immunogenic composition conferscell-mediated immunity against an attenuated mycobacterium that isimmunologically related to M. tuberculosis. The resulting immuneresponse should inhibit primary tuberculosis. Thus, if primarytuberculosis is inhibited, latent infections cannot occur and diseasereactivation is avoided.

Current BCG immunogenic compositions are provided as lyphophilizedcultures that are re-hydrated with sterile diluent immediately beforeadministration. The BCG immunogenic composition is given at birth, ininfancy, or in early childhood in countries that practice BCGvaccination, including developing and developed countries. Adultvisitors to endemic regions who may have been exposed to high doses ofinfectious Mycobacteria may receive BCG as a prophylactic providing theyare skin test non-reactive. Adverse reactions to the immunogeniccomposition are rare and are generally limited to skin ulcerations andlymphadenitis near the injection site. However, in spite of these rareadverse reactions, the BCG immunogenic composition has an unparalleledhistory of safety with over three billion doses having been administeredworldwide since 1930.

However, the unparalleled safety of traditional BCG immunogeniccompositions is coming under increased scrutiny and has created aparadox for healthcare practitioners. The population segments mostsusceptible to mycobacterial infections are the immunosuppressed.Persons suffering from early or late-stage HIV infections areparticularly susceptible to infection. Unfortunately, many persons inthe early-stage of HIV infection are unaware of their immune status. Itis likely that these individuals may voluntarily undergo immunizationusing a live attenuated immunogenic composition such as BCG withoutbeing forewarned of their unique risks. Moreover, other mildlyimmunosuppressed individuals may also unwittingly undergo immunizationwith BCG hoping to avoid mycobacterial disease. Therefore, safer, moreefficacious BCG and BCG-like immunogenic compositions are desirable.

Recently, significant attention has been focused on using transformedBCG strains to produce immunogenic compositions that express variouscell-associated antigens. For example, C. K. Stover, et al. havereported a Lyme Disease immunogenic composition using a recombinant BCG(rBCG) that expresses the membrane associated lipoprotein OspA ofBorrelia burgdorferi. Similarly, the same author has also produced arBCG immunogenic composition expressing a pneumococcal surface protein(PsPA) of Streptococcus pneumoniae. (Stover C K, Bansal G P, LangermanS, and Hanson M S. 1994. Protective immunity elicited by rBCGimmunogenic compositions. In: Brown F. (ed): Recombinant Vectors inImmunogenic composition Development. Dev Biol Stand. Dasel, Karger, Vol.82:163-170)

U.S. Pat. No. 5,504,005 (the “005” patent”) and U.S. Pat. No. 5,854,055(the “055 patent”) both issued to B. R. Bloom et al., disclosetheoretical rBCG vectors expressing a wide range of cell-associatedfusion proteins from numerous species of microorganisms. The theoreticalvectors described in these patents are either directed to cellassociated fusion proteins, as opposed to extracellular non-fusionprotein antigens, and/or the rBCG is hypothetically expressing fusionproteins from distantly related species. Moreover, the recombinantcell-associated fusion proteins expressed in these models are encoded onDNA that is integrated into the host genome and under the control ofheat shock promoters. Consequently, the antigens expressed are fusionproteins and expression is limited to levels approximately equal to, orless than, the vector's native proteins.

Furthermore, neither the '005 nor the '055 patent disclose animal modelsafety testing, immune response development or protective immunity in ananimal system that closely emulates human disease. In addition, onlytheoretical rBCG vectors expressing M. tuberculosis fusion proteins aredisclosed in the '005 and '055 patents; no actual immunogeniccompositions are enabled. Those immunogenic composition models for M.tuberculosis that are disclosed are directed to cell-associated heatshock fusion proteins, not extracellular non-fusion proteins.

U.S. Pat. No. 5,830,475 (the “475 patent”) also discloses theoreticalmycobacterial immunogenic compositions used to express fusion proteins.The DNA encoding these fusion proteins resides in extrachromosomalplasmids under the control of mycobacterial heat shock protein andstress protein promoters. The immunogenic compositions disclosed areintended to elicit immune responses in non-human animals for the purposeof producing antibodies thereto and not shown to prevent intracellularpathogen diseases in mammals. Moreover, the '475 patent does notdisclose recombinant immunogenic compositions that use protein specificpromoters to express extracellular non-fusion proteins.

U.S. Pat. No. 6,467,967 issued to the present inventor, claimsimmunogenic compositions comprising a recombinant BCG having anextrachromosomal nucleic acid sequence comprising a gene encoding a M.tuberculosis 30 kDa major extracellular protein, wherein the M.tuberculosis 30 kDa major extracellular protein is over-expressed andsecreted. Moreover, U.S. Pat. No. 6,924,118 claims additionalrecombinant BCG that over-express other M. tuberculosis majorextracellular proteins.

Therefore, there remains a need for intracellular pathogen immunogeniccompositions that can be safely administered to immunosuppressed, orpartially immunosuppressed, individuals.

SUMMARY OF THE INVENTION

The present invention provides methods for producing recombinantimmunogenic compositions for preventing or treating tuberculosis inhumans and animals, immunogenic compositions against tuberculosis inhumans and animals, and a new approach to producing immunogeniccomposition against tuberculosis, leprosy, other mycobacterial diseases,and other intracellular pathogens.

The present invention provides recombinant BCG immunogenic compositionsthat a) are growth-limited and/or growth-regulatable and b) aregrowth-limited and/or growth-regulatable and secrete a majorextracellular protein of an intracellular pathogen, in one non-limitingexample, a Mycobacteria. tuberculosis major extracellular protein.

In one embodiment of the present invention, An immunogenic compositioncomprising a growth regulatable recombinant Bacille Calmette Guérin(rBCG) having a extrachromosomal nucleic acid sequence comprising a geneencoding for at least one Mycobacteria major extracellular proteinselected from the group consisting of 12 kDa protein, 14 kDa protein, 16kDa protein, 23.5 kDa protein, 24 kDa protein, 30 kDa protein, 32A kDaprotein, 32B kDa protein, 45 kDa protein, 58 kDa protein, 71 kDaprotein, 80 kDa protein, and 110 KD protein, and combinations thereofand combinations thereof, and wherein the Mycobacteria majorextracellular proteins are over expressed and secreted.

In another embodiment, the immunogenic composition further comprises asecond extrachromosomal nucleic acid sequence comprising a gene encodingfor a second Mycobacteria major extracellular protein selected from thegroup consisting of 30 kDa, protein 23.5 kDa protein, 32A kDa proteinand 32B kDa protein, and combinations thereof.

In another embodiment of the immunogenic compositions of the presentinvention, the first extrachromosomal nucleic acid sequence is under thecontrol of a promoter that is not a heat shock promoter or a stressprotein promoter. In another embodiment, the second extrachromosomalnucleic acid sequence is under the control of a promoter that is not aheat shock promoter or a stress protein promoter.

In yet another embodiment of the immunogenic compositions of the presentinvention, the major extracellular proteins are non-fusion proteins.

In another embodiment, the Mycobacteria major extracellular protein isthe 30 kDa protein.

In another embodiment of the immunogenic compositions of the presentinvention, the growth regulatable rBCG is selected from the groupconsisting of prototrophs, auxotrophs and metabolically impaired mutantsand combinations thereof.

In another embodiment of the immunogenic compositions of the presentinvention, the metabolically impaired mutant is a siderophore mutant. Inanother embodiment, the siderophore is a mycobactin or an exochelin.

In another embodiment of the immunogenic compositions of the presentinvention, the growth regulatable rBCG is an auxotroph and whereinpantothenic acid is used to regulate growth of said auxotroph.

The immunogenic composition according to claim 1 wherein saidMycobacteria major extracellular protein is from a Mycobacteria speciesselected from the group consisting of Mycobacterium tuberculosis,Mycobacterium bovis and Mycobacterium leprae.

The immunogenic composition according to claim 11 wherein saidMycobacteria major extracellular protein is a Mycobacterium tuberculosismajor extracellular protein.

In one embodiment of the immunogenic compositions of the presentinvention, an immunogenic composition is provided comprising a rBCGhaving an extrachromosomal nucleic acid sequence comprising a geneencoding for a Mycobacteria major extracellular protein selected fromthe group consisting of 30 kDa, 23.5 kDa, 32 kDa and combinationsthereof, wherein the Mycobacteria major extracellular proteins are overexpressed and secreted; wherein the rBCG is an auxotroph; and whereinpantothenic acid is used to regulate growth of the auxotroph.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts expression and secretion of the M. tuberculosis 30 kDamajor secretory protein by rBCG(panCD)30 (pNBV1-30) Tice [Abbreviated asrBCG(panCD)30] and rBCG(mbtB)30 II (pNBV1-30) Tice [Abbreviated asrBCG(mbtB)30] according to the teachings of the present invention.

FIG. 2 depicts the growth of rBCG(panCD) in THP-1 cells in the presenceand absence of pantothenate according to the teachings of the presentinvention.

FIG. 3 depicts the growth of rBCG(mbtB)30 in broth culture in thepresence of different concentrations of mycobactin J according to theteachings of the present invention.

FIG. 4 depicts the residual growth of rBCG(mbtB)30 in broth culturelacking mycobactin J after growth in the presence of differentconcentrations of mycobactin J according to the teachings of the presentinvention.

FIG. 5 depicts the intracellular growth of rBCG(mbtB)30 in THP-1 cellsin the presence of different concentrations of mycobactin J according tothe teachings of the present invention.

FIG. 6 depicts the residual intracellular growth of rBCG(mbtB)30 inTHP-1 cells in the absence of mycobactin J after growth of rBCG(mbtB)30in broth in the presence of different concentrations of mycobactin Jaccording to the teachings of the present invention.

FIG. 7 depicts the proliferation of splenic lymphocytes from guinea pigsimmunized with BCG, rBCG30, or various auxotrophic strains ofrecombinant BCG in response to the M. tuberculosis 30 kDa majorsecretory protein or Purified Protein Derivative (PPD) according to theteachings of the present invention.

FIG. 8 depicts proliferation of splenic lymphocytes of guinea pigsimmunized with BCG, rBCG30, or various auxotrophic or growth-restrictedstrains of recombinant BCG in response to the M. tuberculosis 30 kDamajor secretory protein or PPD according to the teachings of the presentinvention.

FIG. 9 depicts replication of rBCG(mbtB)30 in guinea pigs according tothe teachings of the present invention.

FIG. 10 depicts replication of rBCG(panCD)30 in guinea pigs according tothe teachings of the present invention.

FIG. 11 depicts the survival of SCID mice infected with rBCG(mbtB)30according to the teachings of the present invention.

FIG. 12 depicts the survival of SCID mice infected with rBCG(panCD)30according to the teachings of the present invention.

DEFINITION OF TERMS

To facilitate an understanding of the following Detailed Description,Examples and appended claims it may be useful to refer to the followingdefinitions. These definitions are non-limiting in nature and aresupplied merely as a convenience to the reader.

Auxotroph or auxotrophic: As used herein “auxotroph” refers to amicroorganism having a specific nutritional requirement not required bythe wild-type organism. In the absence of the required nutrient theauxotroph will not grow whereas the wild-type will thrive.

Gene: A “gene” as used herein refers to at least a portion of a geneticconstruct having a promoter and/or other regulatory sequences requiredfor, or that modify the expression of, the genetic construct.

Genetic Construct: A “genetic construct” as used herein shall mean anucleic acid sequence encoding for at least one major extracellularprotein from at least one intracellular pathogen. In one embodiment ofthe present invention the genetic construct is extrachromosomal DNA.

Growth Regulatable: As used herein the term “growth regulatable” refersto an auxotrophic or metabolically impaired form of the presentinvention's immunogenic compositions. Growth is regulated by providing anutrient essential for the auxotroph's growth at a concentrationsufficient to induce growth.

Host: As used herein “host” refers to the recipient of the presentimmunogenic compositions. Exemplary hosts are mammals including, but notlimited to, primates, rodents, cows, horses, dogs, cats, sheep, goats,pigs and elephants. In one embodiment of the present invention the hostis a human. For the purposes of this disclosure host is synonymous with“vaccinee.”

Immunogen: As used herein the term “immunogen” shall mean any substratethat elicits an immune response in a host. Immunogens of the presentinvention include, but are not limited to major extracellular proteins,and their recombinant forms, derived from intracellular pathogens, suchas, but not limited members of the genus Mycobacterium.

Immunogenic Composition: An “immunogenic composition” as used hereincomprises a recombinant vector, with or without an adjuvant, such as anintracellular pathogen, that expresses and/or secretes an immunogen invivo and wherein the immunogen elicits an immune response in the host.The immunogenic compositions disclosed herein may be prototrophic,auxotrophic or metabolically impaired transformants. The immunogeniccompositions of the present invention may or may not be immunoprotectiveor therapeutic. When the immunogenic compositions of the presentinvention prevent, ameliorate, palliate or eliminate disease from thehost then the immunogenic composition may optionally be referred to as avaccine. However, the term immunogenic composition is not intended to belimited to vaccines.

Major extracellular protein: As used herein, the term “majorextracellular protein” is synonymous with “major secretory protein.” Thepresent inventors have previously described and characterized themycobacterial major extracellular proteins of the present invention. Thedescriptions and characterization of the present major extracellularproteins can be found, without limitation, in U.S. Pat. No. 6,599,510,issued Jul. 29, 2003 to the present inventors, the entire contents ofwhich are hereby incorporated by reference.

Metabolically impaired: As used herein “metabolically impaired” shallmean a recombinant expression vector, specifically a recombinant BacilleCalmette Guérin (rBCG), that has an altered or deleted gene that isessential for normal metabolism. In the present case, the metabolicalteration results in a rBCG that cannot divide in vivo unless thenutrient is provided to the rBCG (pre-loading) prior to the rBCG beingadministered in vivo.

Nucleic Acid Sequence: As used herein the term “nucleic acid sequence”shall mean any continuous sequence of nucleic acids.

Prototrophic: As used herein “prototrophic” refers to a rBCG that thatdoes not require any substance in its nutrition additional to thoserequired by the wild-type.

Transformant: As used herein a “transformant” refers to a microorganismthat has been transformed with at least one heterologous or homologousnucleic acid encoding for a polypeptide that is expressed and/orsecreted. In one embodiment of the present invention the transformant isBCG.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for producing recombinantimmunogenic compositions for preventing or treating tuberculosis inhumans and animals, immunogenic compositions against tuberculosis inhumans and animals, and a new approach to producing immunogeniccomposition against tuberculosis, leprosy, other mycobacterial diseases,and other intracellular pathogens.

Embodiments of the present invention are useful for preventing infectioncaused by Mycobacterium tuberculosis, the agent of tuberculosis,infection by other pathogenic strains of Mycobacteria in humans and/oranimals including Mycobacterium bovis and Mycobacterium leprae; andinfection by intracellular pathogens in general.

A safe and effective vaccine against M. tuberculosis that is more potentthan the currently available vaccine is sorely needed. The onlycurrently available vaccine, Mycobacterium bovis strain Bacille CalmetteGuérin (BCG), is of variable efficacy. Many studies have failed todemonstrate significant protection. One large carefully conductedmeta-analysis has estimated the potency of BCG to be approximately 50%.Hence, a vaccine that improved the potency of BCG by even a small amountcould have a tremendous impact on disease incidence.

The present inventors have previously disclosed recombinant BCGimmunogenic compositions (rBCG30) expressing and secreting the M.tuberculosis 30 kDa major secretory protein (Horwitz et al. Proc. Natl.Acad. Sci. USA 97:113853-13858, 2000). These immunogenic compositionswere more potent than BCG in the highly relevant guinea pig model. Oneof these immunogenic compositions, rBCG30 Tice I (pSMT3-MTB30) is usedin the studies described below.

The present invention provides recombinant BCG immunogenic compositionsthat a) are growth-limited and/or growth-regulatable and b) aregrowth-limited and/or growth-regulatable and secrete a M. tuberculosismajor extracellular protein, in one non-limiting example, the M.tuberculosis 30 kDa major secretory protein.

Growth regulatable recombinant BCG immunogenic compositions, which cannot grow in the host without a nutritional supplement, are designed tobe safer than BCG, because unlike BCG, such immunogenic composition cannot disseminate in the host in the absence of a nutritional supplement.In the present application, growth-regulatable recombinant BCGimmunogenic compositions are provided that are dependent upon smallamounts of the vitamin pantothenate, which can be given safely andinexpensively to mammals in large quantities, facilitating itsacquisition by the live recombinant immunogenic composition in the host.One version of the novel live recombinant pantothenate-dependent BCGimmunogenic composition over-expresses the M. tuberculosis 30 kDa majorsecretory protein.

One embodiment of the present invention provides a growth-limited(growth-restricted) immunogenic composition over-expressing the M.tuberculosis 30 kDa major secretory protein [rBCG(mbtB)30] that lacksthe ability to acquire iron and therefore can not divide more than a fewtimes in the host. Such an immunogenic composition also would be safe inthe immunocompromised host. The rBCG(mbtB)30 immunogenic composition,while safer than BCG because it can not disseminate in animmunocompromised host, is also more potent than BCG.

Embodiments of the present invention therefore provide recombinantstrains of BCG Tice that are growth-limited and/or growth-regulatableincluding strains that secrete pathogen major extracellular proteinsincluding M. tuberculosis major extracellular proteins.

The immunogenic compositions of the present invention are administeredintradermally or by another route, e.g. subcutaneously, intranasally,inhaled, or even orally to a mammalian host. The immunogeniccompositions are suitable for both immunocompetent and immunocompromisedhosts. The immunogenic compositions induce a strong cell-mediated immuneresponse to pathogen antigens in the vaccine. The immunogeniccompositions subsequently protect the mammalian hosts against infectionwith M. tuberculosis, Mycobacterium leprae, Mycobacterium avium, otherMycobacteria, and other intracellular pathogens.

Additionally, the current commercially available BCG vaccine againsttuberculosis is of limited efficacy against pulmonary tuberculosis. Theimmunogenic compositions of the present invention are more potent thanthe current commercially available vaccine in protecting againstpulmonary tuberculosis and dissemination of bacteria to the spleen andother organs. Additionally, the immunogenic compositions of the presentinvention are safer than the current commercially available vaccine inthat the immunogenic compositions are unable to disseminate in theimmunocompromised host.

In one embodiment, the immunogenic compositions use extrachromosomalnucleic acids to express at least one recombinant immunogenic antigengene and placing this gene(s) under the control of non-heat shock genepromoters or non-stress protein gene promoters, preferablyprotein-specific promoter sequences. Consequently, immunogeniccompositions are provided having non-fusion, recombinant immunogenicantigens expressed in greater quantities than possible when genesencoding for recombinant immunogenic antigens are stably integrated intothe immunogenic composition's genomic DNA. As a result, intracellularpathogen immunogenic compositions having surprisingly superiorspecificity and potency than existing subunit or attenuatedintracellular pathogen immunogenic compositions are provided.

The technology described herein for enhancing the immune response of thehost is applicable to other vaccines against intracellular pathogenssuch as vaccines or immunogenic compositions against Francisellatularensis, Chlamydia species, Listeria monocytogenes, Brucella species,Yersinia pestis, Salmonella typhi, Leishmania species, Mycobacteriaspecies, Trypanosoma cruzi, Toxoplasma gondii, Histoplasma capsulatum,Riskettsia species, Coxiella burnetii, Plasmodia species that causemalaria, and Human Immunodeficiency Virus (HIV).

Furthermore, the recombinant immunogenic antigens overexpressed by theimmunogenic compositions disclosed herein can be from species including,but not limited to, Mycobacterium bovis, M. tuberculosis, M. leprae, M.kansasii, M. avium, Mycobacterium sp., Legionella pneumophila, L.longbeachae, L. bozemanii, Legionella sp., Rickettsia rickettsii,Rickettsia typhi, Rickettsia sp., Ehrlichia chaffeensis, Ehrlichiaphagocytophila geno group, Ehrlichia sp., Coxiella burnetii, Leishmaniasp, Toxpolasma gondii, Trypanosoma cruzi, Chlamydia pneumoniae,Chlamydia sp, Listeria monocytogenes, Listeria sp, and Histoplasma sp.

Suitable recombinant immunogenic antigens include the majorextracellular proteins of Mycobacteria species including, but notlimited to, the 12 kDa protein, 14 kDa protein, 16 kDa protein, 23 kDaprotein, 23.5 kDa protein, 30 kDa protein, 32A kDa protein, 32B kDaprotein, 45 kDa protein, 58 kDa protein, 71 kDa protein, 80 kDa protein,and 110 kDa protein and combinations thereof.

1. BCG Mutant Defective in Pantothenate Biosynthesis

rBCG(panCD) Tice: A BCG mutant defective in pantothenate biosynthesis(pantothenate auxotroph) was constructed by disrupting the BCG TicepanCD genes via allelic exchange. The allelic exchange substrate wasgenerated using a cloning strategy in which a panCD locus with a 1.3 kbdeletion was created with an apramycin resistance (apr^(r)) geneinserted at the site of the deletion. This mutated allele was clonedinto the allelic exchange vector phEX1 (a derivative of phAE87 [Bardarovet al., Microbiol. 148:3007-3017, 2002]) to generate phEX1ΔpanCD::apr^(r). This plasmid was electroporated into Mycobacteriumsmegmatis to generate the specialized transducing phage. BCG Tice wasinfected with this purified phage and then plated on 7H10 platescontaining 50 μg mL⁻¹ apramycin and 50 μg mL⁻¹ calcium D-pantothenate toselect for clones that had undergone a homologous recombination event.Four apramycin resistant clones were obtained of which two were shown tobe pantothenate auxotrophs. No growth was observed for the auxotrophs onplates or in broth without the addition of calcium D-pantothenate. Inbroth culture, the mutant strain grows at a rate similar to thewild-type strain in the presence of ≧10 μg mL⁻¹ calcium D-pantothenate.To ensure a pure culture, one of the pantothenate auxotrophic clones wasplated at low density and a single colony was reisolated. The strainname is abbreviated as rBCG(panCD).

2. BCG Mutant Defective in Pantothenate Biosynthesis Expressing andSecreting the M. tuberculosis 30 kDa Major Secretory Protein

rBCG(panCD)30 (pNBV1-30) Tice: A BCG mutant defective in pantothenatebiosynthesis that also expresses and secretes the M. tuberculosis 30 kDamajor secretory protein was constructed by electroporating the plasmidpNBV1-30 (hyg^(r)) into rBCG(panCD) and selecting transformants on 7H11agar with 50 μg mL⁻¹ hygromycin, 50 μg mL⁻¹ apramycin, and 50 μg mL⁻¹pantothenate. The plasmid pNBV1-30 was constructed by cloningapproximately 1.5 kb of DNA containing the coding region of the M.tuberculosis 30 kDa major secretory protein gene (fbpB) andapproximately 500 by of sequence upstream of the start codon into themulti-cloning site of pNBV1 (Howard et al. Geme 166:181-182, 1995). Asingle hygromycin and apramycin resistant clone was cultured in 7H9medium containing 0.01% (w/v) tyloxapol, 50 μg mL⁻¹ hygromycin, and 50μg mL⁻¹ pantothenate. The clone was found to express and export a highlevel of recombinant M. tuberculosis 30 kDa major secretory protein asdetermined by polyacrylamide gel electrophoresis (FIG. 1). The strainstably expressed and exported the 30 kDa major secretory protein even inthe absence of the selective antibiotic hygromycin for at least ˜40generations (4 subcultures, 1:1000 dilutions). The strain name isabbreviated as rBCG(panCD)30.

3. BCG Mutant Defective in Iron Acquisition Expressing and Secreting theM. tuberculosis 30 kDa Major Secretory Protein

rBCG(mbtB)30 II (pNBV1-30) Tice: A BCG mutant defective in ironacquisition that also expresses and secretes the M. tuberculosis 30 kDamajor secretory protein was constructed by electroporating the plasmidpNBV1-30 (hyg^(r)) into rBCG(mbtB) (also known as rBCG-mbtB) andselecting transformants on 7H10 agar with 50 μg mL⁻¹ hygromycin and 1 μgmL⁻¹ mycobactin J. Eight individual hygromycin resistant clones wererandomly selected and screened for expression and export of recombinantM. tuberculosis 30 kDa major secretory protein by polyacrylamide gelelectrophoresis. All eight clones were found to express and export ahigh level of the 30 kDa protein. The strain stably expressed andexported the 30 kDa major secretory protein even in the absence of theselective antibiotic hygromycin for at least ˜50 generations (5subcultures, 1:1000 dilutions). One such clone is shown in FIG. 1. Thestrain name is abbreviated as rBCG(mbtB)30.

4. BCG Mutant Defective in L-Tryptophan Biosynthesis Expressing andSecreting the M. tuberculosis 30 kDa Major Secretory Protein

rBCG(trpD)30 (pSMT3-MTB30) Tice: A BCG mutant defective in L-tryptophanbiosynthesis that also expresses and secretes the M. tuberculosis 30 kDamajor secretory protein was constructed by electroporating the plasmidpSMT3-MTB30 (hyg^(r)) into the L-trypthophan auxotroph strain rBCG(trpD)(also known as BCG Tice trpD) and selecting transformants on 7H11 agarwith 50 μg mL⁻¹ hygromycin, 50 μg mL⁻¹ kanamycin, and 50 μg mL⁻¹L-tryptophan. Ten individual hygromycin and kanamycin resistant cloneswere randomly selected and screened for expression and export ofrecombinant M. tuberculosis 30 kDa major secretory protein bypolyacrylamide gel electrophoresis by polyacrylamide gel electrophoresisand immunoblotting with polyvalent, highly specific rabbit anti-30 kDaprotein immunoglobulin. The strain was found to produce ˜10-20 timesmore 30 kDa antigen per mL of culture than a control BCG Tice strain.The strain name is abbreviated as rBCG(trpD)30.

Example 1 Studies of Growth of Auxotrophic Strains in vitro: rBCG(panCD)and rBCG(panCD)30 (pNBV1-30) Tice

The rBCG(panCD) and rBCG(panCD)30 strains are pantothenate (Vitamin B5)auxotrophs. In broth culture, rBCG(panCD) grew at a rate similar to thewild-type BCG strain in the presence of ≧10 μg mL⁻¹ pantothenate.rBCG(panCD) displayed less than maximal intracellular growth in humanmacrophages when macrophages were cultured in standard tissue culturemedium which contains 0.25 μg mL⁻¹ pantothenate. Intracellular growthsimilar to the wild-type strain was achieved by adding an additional 100μg mL⁻¹ pantothenate (0.42 mM) to the tissue culture medium (FIG. 2,culture A was grown with 50 μg mL⁻¹ pantothenate and culture B was grownwith 500 μg mL⁻¹ pantothenate prior to infection of the macrophages).

Example 2 Studies of Growth-Restricted Strains In Vitro: rBCG(mbtB) andrBCG(mbtB)30 II (pNBV1-30) Tice

The rBCG(mbtB)30 strain is mycobactin-dependent and thus it is defectivein iron acquisition. Growth of the strain in broth is normal whensupplemented with ≧10 ng/mL mycobactin J while growth is reduced atmycobactin J concentrations ≦1 ng/mL (FIG. 3). Although rBCG(mbtB)30 isdependent on exogenous mycobactin for growth, the strain can multiplyfor a limited number of generations after removal of mycobactin J (FIG.4). The number of generations of residual growth in the absence ofmycobactin is dependent upon the level of mycobactin J in the medium inwhich the strain was initially grown. When initially grown in brothsupplemented with a high concentration of mycobactin J (10 μg/mL) andthe cells washed, the strain is capable of approximately 10 generationsof growth after subculture into medium lacking mycobactin J. Wheninitially grown in broth supplemented with a low concentration ofmycobactin J (0.01 μg/mL) and the cells washed, the strain is capable ofonly approximately 2 generations of growth after subculture into mediumlacking mycobactin J.

The capacity of rBCG(mbtB)30 for residual growth after the removal ofmycobactin J was also tested in human THP-1 macrophages (FIG. 5). Whenthe bacteria were initially grown in a low concentration of mycobactin J(0.01 μg/mL) and then added to THP-1 cells, there was marginal growth inthe absence of mycobactin J. Growth was normal, or nearly so, when thetissue culture medium was supplemented with ≧1 ng/mL of mycobactin J.The residual growth of rBCG(mbtB)30 in the absence of mycobactin J wasalso evaluated in human THP-1 macrophages (FIG. 6). When grown in a lowconcentration of mycobactin J (0.01 μg/mL) prior to infection of THP-1cells, rBCG(mbtB)30 displayed little net growth over the seven daysinfection period (equivalent to approximately 1 generation). When grownin a high concentration of mycobactin J (10 μg/mL) prior to infection ofthe THP-1 cells, rBCG(mbtB)30 multiplied approximately 1 log over thecourse of the infection (equivalent to ˜3 generations).

Example 3 Survival Studies of Auxotrophic and Growth-Restricted StrainsIn Vivo

The studies of the survival of the auxotrophic and growth-restrictedvaccine strains utilized guinea pigs because the guinea pig model isespecially relevant to human tuberculosis. Furthermore, the wild-typeBCG vaccine is known to undergo multiplication in guinea pigs prior tocontainment by the guinea pig's immune system. Multiplication of the BCGvaccine may be required for optimal immunity against tuberculosis.

Aliquots were removed from logarithmically growing wild-type orrecombinant BCG cultures, and the bacteria were pelleted bycentrifugation at 3,500×g for 15 min. The bacteria were then washed with1× phosphate buffered saline (1× PBS, 50 mM sodium phosphate pH 7, 150mM sodium chloride) and resuspended in 1× PBS to the desiredconcentration. The immunization inoculum contained from 10³ to 10⁷viable wild-type or recombinant BCG bacteria in a total volume of 100μl.

Experiment 1

1. Immunization of Animals

Specific-pathogen free 250-300 g outbred male Hartley strain guinea pigsfrom Charles River Breeding Laboratories, in groups of 3 were immunizedintradermally with 10⁶-10⁷ CFU of one of the following strains of BCG:

Group A: BCG Tice Parental Control (BCG)

Group B: rBCG30 Tice I (pSMT3-MTB30)(rBCG30)

Group C: rBCG(trpD) Tice (rBCG(trpD))

Group D: rBCG(trpD)30 (pSMT3-MTB30) Tice (rBCG(trpD)30)

Group E: rBCG(trpD) Tice—Animals fed diet high in tryptophan(rBCG(trpD)-diet)

Group F: rBCG(trpD)30 (pSMT3-MTB30) Tice—Animals fed diet high intryptophan (rBCG(trpD)30-diet)

Group G: rBCG(panCD) Tice (rBCG(panCD))

Group H: rBCG(panCD) Tice—Animals fed diet high in pantothenate(rBCG(panCD)-diet)

Group I: rBCG(mbtB) Tice—Grown in medium containing a low mycobactinconcentration (rBCG(mbtB) Lo Fe)

Group J: rBCG(mbtB) Tice—Grown in medium containing a high mycobactinconcentration (rBCG(mbtB) Hi Fe)

2. Survival of Vaccines in Guinea Pigs

Three weeks after immunization the animals were euthanized and thespleen of each animal was removed and cultured for colony forming units(CFU) of BCG on Middlebrook 7H11 agar containing 50 μg/mL L-tryptophan,50 μg/mL pantothenate, and 0.1 μg/mL mycobactin J for three weeks at 37°C., 5% CO₂-95% air atmosphere. The results of the assay for CFU in thespleens are shown in Table 1. The limit of detection was 4 CFU (0.6 logCFU).

TABLE 1 Vaccine Survival in vivo - Experiment 1 Spleen Group Strain(Mean Log CFU ± SE) A BCG 2.8 ± 0.2 B rBCG30 3.9 ± 0.1 C rBCG(trpD) 0.7± 0.1 D rBCG(trpD)30 0.7 ± 0.0 E rBCG(trpD)-diet 1.3 ± 0.3 FrBCG(trpD)30-diet 0.9 ± 0.2 G rBCG(panCD) 0.7 ± 0.0 H rBCG(panCD)-diet0.9 ± 0.3 I rBCG(mbtB) Lo Fe 1.3 ± 0.4 J rBCG(mbtB) Hi Fe 1.9 ± 0.4

These results showed that animals immunized with BCG and rBCG30 had arelatively high level of BCG organisms (2.8 and 3.9 log CFU) in theirspleens three weeks after immunization, while the animals immunized withthe auxotrophic BCG vaccines had much lower levels of bacteria (0.7-1.3log CFU) in their speens, thus demonstrating a much higher degree ofsafety with these strains. In addition, there was evidence thatsupplementation of the guinea pigs diet with the nutrient required bythe auxotrophic vaccine (L-tryptophan or pantothenate) resulted ingreater survival of the auxotrophic strains (rBCG(trpD): 0.7 log CFU vs.rBCG(trpD)-diet: 1.3 log CFU; rBCG(trpD)30: 0.7 log CFU vs.rBCG(trpD)30-diet: 0.9 log CFU; rBCG(panCD): 0.7 log CFU vs.rBCG(panCD)-diet: 0.9 log CFU). The growth-restricted vaccine,rBCG(mbtB), had greater survival in the spleen than the auxotrophicstrains, but still less than BCG and rBCG30, demonstrating greatersafety. Furthermore, survival was increased by growing the vaccine witha high mycobactin J concentration (iron loaded vaccine) prior toimmunization (rBCG(mbtB) Lo Fe: 1.3 log CFU vs. rBCG(mbtB) Hi Fe: 1.9log CFU) demonstrating that growth and/or survival of thegrowth-restricted strain rBCG(mbtB) can be controlled by changing thelevel of iron loading of the strain before immunization.

Example 4 Cell-Mediated, Humoral, and Protective Immunity Studies

The studies of the efficacy of the vaccines utilized guinea pigs becausethe guinea pig model is especially relevant to human tuberculosisclinically, immunologically, and pathologically. In contrast to themouse and rat, but like the human, the guinea pig a) is susceptible tolow doses of aerosolized M. tuberculosis; b) exhibits strong cutaneousdelayed-type hypersensitivity (DTH) to tuberculin; and c) displaysLanghans giant cells and caseation in pulmonary lesions. However,whereas only about 10% of immunocompetent humans who are infected withM. tuberculosis develop active disease over their lifetime (half earlyafter exposure and half after a period of latency), infected guinea pigsalways develop early active disease. While guinea pigs differ fromhumans in this respect, the consistency with which they develop activedisease after infection with M. tuberculosis is an advantage in trialsof vaccine efficacy.

Aliquots were removed from logarithmically growing wild-type orrecombinant BCG cultures, and the bacteria were pelleted bycentrifugation at 3,500×g for 15 min. The bacteria were then washed with1× phosphate buffered saline (1× PBS, 50 mM sodium phosphate pH 7, 150mM sodium chloride) and resuspended at a final concentration of 1×10⁴ or1×10⁷ colony-forming units per ml in 1×PBS. The immunization inoculumcontained 10³ or 10⁶ viable wild-type or recombinant BCG bacteria in atotal volume of 100 μl.

Experiment 1

1. Immunization of Animals

Specific-pathogen free 250-300 g outbred male Hartley strain guinea pigsfrom Charles River Breeding Laboratories, in groups of 15 or 21, weresham-immunized by intradermal administration of buffer (15 animalstotal) or immunized intradermally with 10³ or 10⁶ CFU of one of thefollowing strains of BCG (21 animals/group):

Group A: Sham-immunized (Sham)

Group B: 10³ BCG Tice Parental Control (BCG)

Group C: 10³ rBCG30 Tice I (pSMT3-MTB30)(rBCG30)

Group D: 10³ rBCG30/hINFγ (pSMT3-MTB30; pGB9.2-hINFγ) Tice(rBCG30/hINFγ)

Group E: 10⁶ rBCG(mbtB) Tice—Grown in medium containing a low mycobactinconcentration (rBCG(mbtB) Lo Fe)

Group F: 10⁶ rBCG(mbtB) Tice—Grown in medium containing a highmycobactin concentration (rBCG(mbtB) Hi Fe)

Group G: 10⁶ rBCG(mbtB)30 II (pNBV1-30) Tice—Grown in medium containinga low mycobactin concentration (rBCG(mbtB)30 Lo Fe)

Group H: 10⁶ rBCG(mbtB)30 II (pNBV1-30) Tice—Grown in medium containinga high mycobactin concentration (rBCG(mbtB)30 Hi Fe)

2. Cutaneous Delayed-type Hypersensitivity (DTH) to Purified RecombinantM. tuberculosis 30 kDa Major Secretory Protein (r30)

Ten weeks after immunization, 6 guinea pigs in each group were shavedover the back and injected intradermally with 10 μg of purifiedrecombinant M. tuberculosis 30 kDa major secretory protein (r30) in 100μl phosphate buffered saline. After 24 h, the diameter of erythema andinduration was measured. (A separate group of animals from the one usedin the challenge studies was used for skin-testing to eliminate thepossibility that the skin test itself might influence the outcome). Theresults are summarized in Table 2.

TABLE 2 Cutaneous DTH - Experiment 1 Erythema Induration Group StrainTest Antigen (mm ± SE) (mm ± SE) A Sham r30 2.7 ± 1.3 0 ± 0 B BCG r305.8 ± 1.4 0 ± 0 C rBCG30 r30 11.8 ± 2.6  5.6 ± 3.5 D rBCG30/hINFγ r306.3 ± 1.4 0 ± 0 E rBCG(mbtB) Lo Fe r30 2.0 ± 1.0 0 ± 0 F rBCG(mbtB) HiFe r30 3.0 ± 1.6 0 ± 0 G rBCG(mbtB)30 r30 14.4 ± 2.3  3.6 ± 3.6 Lo Fe HrBCG(mbtB)30 r30 10.3 ± 1.3  4.7 ± 2.1 Hi Fe

These results showed that sham-immunized animals (Group A) and animalsimmunized with the parental BCG Tice strain (Group B) had no indurationupon testing with r30. Similarly, animals immunized with agrowth-restricted vaccine [rBCG(mbtB)] not over-expressing the 30 kDaprotein had no induration upon testing with r30, whether the vaccine wasgrown under high mycobactin J (Group F) or low mycobactin J (Group E)conditions. In contrast, animals immunized with a recombinant BCG strainoverexpressing r30 (Group C) had induration in response to r30.Similarly, animals immunized with the growth-restricted strainrBCG(mbtB)30, whether grown under high mycobactin J (Group H) or lowmycobactin J (Group G) conditions, showed induration upon testing withr30. Interestingly, the recombinant BCG expressing both r30 and humaninterferon gamma did not show induration upon testing with r30, althoughit did display some erythema.

3. Protective Immunity to Aerosol Challenge.

Ten weeks after immunization, the remaining animals in Groups A-H werechallenged with an aerosol generated from a 10 ml single-cell suspensioncontaining 7.5×10⁴ colony-forming units (CFU) of M. tuberculosis. Priorto challenge, the challenge strain, M. tuberculosis Erdman strain (ATCC35801), had been passaged through outbred guinea pigs to maintainvirulence, cultured on 7H11 agar, subjected to gentle sonication toobtain a single cell suspension, and frozen at −70° C. This aerosol dosedelivered ˜10 live bacilli to the lungs of each animal. The airborneroute of infection was used because this is the natural route ofinfection for pulmonary tuberculosis. A relatively large dose was usedso as to induce measurable clinical illness in 100% of control animalswithin a relatively short time frame (10 weeks). Afterwards, guinea pigswere individually housed in stainless steel cages contained within alaminar flow biohazard safety enclosure and allowed free access tostandard laboratory chow and water. The animals were observed forillness and weighed weekly for 10 weeks and then euthanized. The rightlung and spleen of each animal was removed and cultured for CFU of M.tuberculosis on Middlebrook 7H11 agar for two weeks at 37° C., 5%CO₂-95% air atmosphere. The results of the assay for CFU in the lungsand spleens are shown in Table 3.

TABLE 3 CFU in Lungs and Spleens - Experiment 1 Spleen Lung (Mean LogGroup Strain (Mean Log CFU ± SE) CFU ± SE) A Sham 6.03 ± 0.10 5.57 ±0.17 B BCG 4.83 ± 0.12 4.20 ± 0.21 C rBCG30 4.04 ± 0.21 2.87 ± 0.23 DrBCG30/hINFγ 3.57 ± 0.27 2.38 ± 0.30 E rBCG(mbtB) Lo Fe 4.49 ± 0.19 3.69± 0.33 F rBCG(mbtB) Hi Fe 4.68 ± 0.25 4.42 ± 0.24 G rBCG(mbtB)30 Lo Fe4.40 ± 0.28 3.17 ± 0.39 H rBCG(mbtB)30 Hi Fe 4.42 ± 0.25 2.93 ± 0.31

These results showed that animals immunized with BCG or any recombinantBCG strain had much lower CFU in the lungs and spleens than the shamimmunized animals.

Animals immunized with the recombinant BCG strain secreting both the M.tuberculosis 30 kDa major secretory protein and human interferon gamma(rBCG30/hINFγ) had markedly fewer CFU in the lung and spleen than evenrBCG30; animals immunized with rBCG30/hINFγ had 0.5 logs fewer CFU inthe lung and spleen than rBCG30. Moreover, in the case of animalsimmunized with rBCG30/hINFγ, 50% of the animals had no detectable CFU intheir spleens and thus were scored at the limit of detection of 1.56logs. In contrast, in the case of rBCG30 immunized animals, only 14% ofthe animals had no detectable CFU in the spleen. Compared with animalsimmunized with BCG, animals immunized with rBCG30/hINFγ had 1.3 logsfewer CFU in the lung and 1.8 logs fewer CFU in the spleen.

Furthermore animals immunized with the growth-restricted strainrBCG(mbtB) had fewer CFU in the lung than BCG, whether grown in high orlow concentrations of mycobactin J before immunization. Remarkably,animals immunized with the growth-restricted recombinant BCG strainover-expressing the M. tuberculosis 30 kDa major secretory protein[rBCG(mbtB)30], whether grown in the presence of high or low amounts ofmycobactin J before immunization, showed an impressive reduction in CFUin animal organs compared with BCG. Animals immunized with rBCG(mbtB)30,whether grown in the presence of high or low amounts of iron beforeimmunization, had 0.4 logs fewer CFU in the lungs than BCG-immunizedanimals; animals immunized with rBCG(mbtB)30 grown in a low amount ofmycobactin J before immunization had 1.0 log fewer CFU in the spleen andanimals immunized with rBCGmbtB-30 grown in a high amount of mycobactinJ before immunization had 1.3 log fewer CFU in the spleen thanBCG-immunized animals. Remarkably, the reduction in CFU in the spleen inanimals immunized with rBCG(mbtB)30 grown in a high amount of mycobactinbefore immunization was comparable to that observed with rBCG30, whichis not growth-restricted.

Experiment 2

1. Immunization of Animals

Specific-pathogen free 250-300 g outbred male Hartley strain guinea pigsfrom Charles River Breeding Laboratories, in groups of 6, weresham-immunized by intradermal administration of buffer or immunizedintradermally with 10³ or 10⁶ CFU of one of the following strains ofBCG:

Group A: Sham-immunized (Sham)

Group B: 10³ BCG Tice Parental Control (BCG)

Group C: 10³ rBCG30 Tice I (pSMT3-MTB30) (rBCG30)

Group D: 10³ rBCG30/hINFγ (pSMT3-MTB30; pGB9.2-hINFγ) Tice(rBCG30/hINFγ)

Group E: 10³ rBCG30/hGM-CSF(pSMT3-MTB30; pGB9.2-hGM-CSF) Tice(rBCG30/hGM-CSF)

Group F: 10³ rBCG30/hIL-2 (pSMT3-MTB30; pGB9.2-hIL-2) Tice(rBCG30/hIL-2)

Group G: 10³ rBCG30/hIL-12 (pSMT3-MTB30; pGB9.2-hIL-12) Tice(rBCG30/hIL-12)

Group H: 10³ rBCG(panCD)30 (pNBV1-30) Tice (10³ rBCG(panCD)30)

Group I: 10⁶ rBCG(panCD)30 (pNBV1-30) Tice (10⁶ rBCG(panCD)30)

Group J: 10³ rBCG(panCD)30 (pNBV1-30) Tice—Animals fed diet high inpantothenate (10³ rBCG(panCD)30-diet)

Group K: 10⁶ rBCG(panCD)30 (pNBV1-30) Tice—Animals fed diet high inpantothenate (10⁶ rBCG(panCD)30-diet)

2. Cutaneous Delayed-type Hypersensitivity (DTH) to Purified RecombinantM. tuberculosis 30 kDa Major Secretory Protein (r30)

Five weeks after immunization, 6 guinea pigs in each group were shavedover the back and injected intradermally with 10 μg of purifiedrecombinant M. tuberculosis 30 kDa major secretory protein (r30) in 100μl phosphate buffered saline. After 24 h, the diameter of erythema andinduration was measured. (A separate group of animals from the one usedin the challenge studies—see below—was used for skin-testing toeliminate the possibility that the skin-test itself might influence theoutcome). The results are summarized in Table 4.

TABLE 4 Cutaneous DTH - Experiment 2 Test Erythema Induration GroupStrain Antigen (mm ± SE) (mm ± SE) A Sham r30 0 ± 0 0 ± 0 B BCG r30 0 ±0 0 ± 0 C rBCG30 r30 16.5 ± 1.6  14.0 ± 3.2  D rBCG30/hINFγ r30 6.8 ±1.5 1.2 ± 1.2 E rBCG30/hGM-CSF r30 6.3 ± 1.6 3.0 ± 1.9 F rBCG30/hIL-2r30 13.5 ± 3.2  13.5 ± 3.2  G rBCG30/hIL-12 r30 5.7 ± 1.9 4.3 ± 2.1 H10³ rBCG(panCD)30 r30 4.3 ± 1.6 0 ± 0 I 10⁶ rBCG(panCD)30 r30 16.1 ±1.1  16.3 ± 1.0  J 10³ rBCG(panCD)30-diet r30 5.8 ± 1.4 0 ± 0 K 10⁶rBCG(panCD)30-diet r30 15.2 ± 0.8  13.0 ± 2.7 

These results showed that sham-immunized animals (Group A) and animalsimmunized with the parental BCG Tice strain (Group B) had no erythema orinduration upon testing with r30. In contrast, animals immunized withrBCG30 or recombinant BCG strains producing both r30 and a humancytokine, displayed erythema and induration in response to skin-testing.Animals immunized with a high dose of rBCG(panCD)30 requiringpantothenate for growth displayed marked erythema and indurationcomparable to that of rBCG30. Animals immunized with a low dose ofrBCG(panCD)30 requiring pantothenate for growth displayed some erythemabut no induration. Interestingly, whether the animals were fed a high orstandard amount of pantothenate in their diet did not significantlyinfluence the amount of induration at a given dose of vaccine. Thus,animals immunized with the new strains secreting the 30 kDa majorsecretory protein in combination with a human immunostimulatory cytokinedeveloped a cell-mediated immune response to r30. In addition, animalsimmunized with a high vaccine dose of rBCG(panCD)30 developed acell-mediated immune response to r30.

3. Antibody to Purified Recombinant M. tuberculosis 30 kDa Major Protein(r30)

Blood was obtained from the animals described above immediately afterthey were euthanized, and the serum was assayed for antibody titer tor30 by ELISA, using Costar (Corning, N.Y.) 96-well EIA/RIA High BindingPlates, r30 at 1 μg/well, guinea pig serum diluted 1:64 to 1:1,024,000,alkaline phosphatase-conjugated goat anti-guinea pig IgG (Sigma, St.Louis, Mo.) at a dilution of 1:1,000, and an Alkaline PhosphataseSubstrate Kit (BioRad, Hercules, Calif.). Titers of less than 1:64 werescored as 32 for statistical purposes. The results are summarized inTable 5.

TABLE 5 Antibody to r30 - Experiment 2 Group Strain Test AntigenGeometric Mean Titer A Sham r30 84 B BCG r30 127 C rBCG30 r30 1154 DrBCG30/hINFγ r30 48 E rBCG30/hGM-CSF r30 73 F rBCG30/hIL-2 r30 49 GrBCG30/hIL-12 r30 37 H 10³ rBCG(panCD)30 r30 32 I 10⁶ rBCG(panCD)30 r30110 J 10³ rBCG(panCD)30-diet r30 37 K 10⁶ rBCG(panCD)30-diet r30 574

These results showed that sham-immunized animals (Group A) and animalsimmunized with the parental BCG Tice strain (Group B) had relatively lowantibody titers to r30. In contrast, animals immunized with rBCG30 had arelatively high titer. Interestingly, animals immunized with recombinantBCG expressing r30 and a cytokine had low titers, indicating that thepresence of the cytokine resulted in a diminished antibody response.

Animals immunized with a low dose of rBCG(panCD)30 requiringpantothenate for growth had low titers with or without dietarysupplementation with pantothenate. Animals immunized with a high dose ofrBCG(panCD)30 requiring pantothenate for growth had a slightly higherantibody titer in the absence of pantothenate dietary supplementationthan animals immunized with a low dose of this strain. However, theantibody titer was markedly increased in animals immunized with a highdose of rBCG(panCD)30 requiring pantothenate for growth who were fed adiet rich in pantothenate. The higher titer is consistent with increasedsurvival of the mutant strain in vivo in animals fed pantothenate.

Experiment 3

1. Immunization of Animals

Specific-pathogen free 250-300 g outbred male Hartley strain guinea pigsfrom Charles River Breeding Laboratories, in groups of 3 or 6, wereimmunized intradermally with 10³ or 10⁶ CFU of one of the followingstrains:

Group B: 10³ BCG Tice Parental Control (BCG) (BCG)

Group C: 10³ rBCG30 Tice I (pSMT3-MTB30) (rBCG30)

Group H: 10³ rBCG(panCD)30 (pNBV1-30) Tice (10³ rBCG(panCD)30)

Group I: 10⁶ rBCG(panCD)30 (pNBV1-30) Tice (10⁶ rBCG(panCD)30)

Group J: 10³ rBCG(panCD)30 (pNBV1-30) Tice—Animals fed diet high inpantothenate(10³ rBCG(panCD)30-diet)

Group K: 10⁶ rBCG(panCD)30 (pNBV1-30) Tice—Animals fed diet high inpantothenate(10⁶ rBCG(panCD)30-diet)

2. Lymphocyte Proliferation

Three weeks after immunization, the animals were euthanized and thespleen was removed for lymphocyte proliferation studies. Spleniclymphocytes were purified as described (Pal and Horwitz, Infect. Immun.60:4781-4792, 1992) and incubated at a final concentration of 10⁷/ml inRPMI1640 containing 12.5 mM HEPES, penicillin (100 U/ml), streptomycin(100 μg/ml), polymyxin B sulfate (100 Units/ml), and 10% fetal calfserum (Gibco) with PPD (10 μg/ml) or with 100, 10, or 1 μg/ml ofpurified M. tuberculosis 30 kDa major secretory protein (r30) in a totalvolume of 100 μl in microtest wells (96-well round-bottom tissue cultureplate; Falcon Labware, Oxnard, Calif.) for 2 days at 37° C. in 5%CO₂-95% air and 100% humidity. As negative and positive controls,lymphocytes were incubated with buffer only (RPMI) or with concanavalinA (15 μg/ml). Subsequently, [³H]thymidine incorporation was determinedand mean Counts Per Minute (CPM) calculated. Stimulation Indices (SI)were calculated using the following formula: SI=CPM with Antigen/CPMwithout Antigen. The results are shown in FIG. 7.

Lymphocytes from animals immunized with BCG had a weak proliferativeresponse to r30, but a moderately strong response to PPD. In contrast,lymphocytes from animals immunized with rBCG30 had a strongproliferative response to both r30 and PPD. Lymphocytes from animalsimmunized with a low dose of rBCG(panCD)30 had a poor proliferativeresponse to both r30 and PPD; however, lymphocytes from animalsimmunized with a high dose of rBCG(panCD)30 had a strong proliferativeresponse to both r30 and PPD. When animals immunized with a low dose ofrBCG(panCD)30 were fed a high pantothenate diet, lymphocytes from theseanimals had an increased proliferative response. Similarly, when animalsimmunized with a high dose of rBCG(panCD)30 were fed a high pantothenatediet, lymphocytes from these animals showed an increased proliferativeresponse.

Experiment 4

1. Immunization of Animals

Specific-pathogen free 250-300 g outbred male Hartley strain guinea pigsfrom Charles River Breeding Laboratories, in groups of 3 were immunizedintradermally with 10⁶-10⁷ CFU of one of the following strains:

Group A: BCG Tice Parental Control (BCG)

Group B: rBCG30 Tice I (pSMT3-MTB30) (rBCG30)

Group C: rBCG(trpD) Tice (rBCG(trpD))

Group D: rBCG(trpD)30 (pSMT3-MTB30) Tice (rBCG(trpD)30)

Group E: rBCG(trpD) Tice—Animals fed diet high in tryptophan(rBCG(trpD)-diet)

Group F: rBCG(trpD)30 (pSMT3-MTB30) Tice—Animals fed diet high intryptophan (rBCG(trpD)30-diet)

Group G: rBCG(panCD) Tice (rBCG(panCD))

Group H: rBCG(panCD) Tice—Animals fed diet high in pantothenate(rBCG(panCD)-diet)

Group I: rBCG(mbtB) Tice—Grown in medium containing a low mycobactin Jconcentration (rBCG(mbtB) Lo Fe)

Group J: rBCG(mbtB) Tice—Grown in medium containing a high mycobactin Jconcentration (rBCG(mbtB) Hi Fe)

2. Lymphocyte Proliferation

Three weeks after immunization, the animals were euthanized and thespleen was removed for lymphocyte proliferation studies. Spleniclymphocytes were purified and incubated at a final concentration of10⁷/ml in RPMI1640 containing 12.5 mM HEPES, penicillin (100 U/ml),streptomycin (100 mg/ml), polymyxin B sulfate (100 Units/ml), and 10%fetal calf serum with PPD (10 μg/ml) or with 100 μg/ml of purified M.tuberculosis 30 kDa major secretory protein (r30) in a total volume of100 μl in microtest wells for 2 days at 37° C. in 5% CO₂-95% air 100%humidity. As negative and positive controls, lymphocytes were incubatedwith buffer only (RPMI) or with concanavalin A (15 82 g/ml).Subsequently, [³H]thymidine incorporation was determined and mean CPMcalculated. Stimulation Indices (SI) were calculated using the followingformula: SI=CPM with Antigen/CPM without Antigen. The results are shownin FIG. 8.

Lymphocytes from animals immunized with BCG had a weak proliferativeresponse to r30, but a moderately strong response to PPD. In contrast,lymphocytes from animals immunized with rBCG30 had a strongproliferative response to both r30 and PPD. Lymphocytes from animalsimmunized with rBCG(trpD) or rBCG(trpD)30 responded similarly to BCG.When animals immunized with rBCG(trpD) were fed a high tryptophan diet,lymphocytes from these animals showed a response similar to lymphocytesfrom animals immunized with rBCG(trpD) and not fed a high tryptophandiet. However, when animals immunized with rBCG(trpD)30 were fed a hightryptophan diet, lymphocytes from these animals showed an increasedproliferative response compared with lymphocytes from animals immunizedwith rBCG(trpD)30 and not fed a high tryptophan diet. Lymphocytes fromanimals immunized with rBCG(panCD) showed a modest response to r30 and amoderately strong response to PPD. When animals immunized withrBCG(panCD) were fed a high pantothenate diet, lymphocyte proliferativeresponses to r30 and PPD did not increase but instead somewhatdecreased. Lymphocytes from animals immunized with rBCG(mbtB) grown inthe presence of low mycobactin J proliferated moderately strongly toboth r30 and PPD. Similar lymphocyte proliferative responses were seenin lymphocytes from animals immunized with rBCG(mbtB) grown in thepresence of a high level of mycobactin J.

Experiment 5

1. Immunization of Animals

Specific-pathogen free 250-300 g outbred male Hartley strain guinea pigsfrom Charles River Breeding Laboratories, in groups of 15 or 21, weresham-immunized by intradermal administration of buffer (15 animalstotal) or immunized intradermally with 10³ or 10⁶ CFU of one of thefollowing strains (21 animals/group):

Group A: Sham-immunized (Sham)

Group B: 10³ BCG Tice Parental Control (BCG)

Group C: 10³ rBCG30 Tice I (pSMT3-MTB30) (rBCG30)

Group D: 10³ rBCG30/hINFγ (pSMT3-MTB30; pGB9.2-hINFγ) Tice(rBCG30/hINFγ)

Group E: 10³ rBCG/hINFγ (pGB9.2-hINFγ) Tice (rBCG/hINFγ)

Group F: 10³ BCG Tice Parental Control—Grown in medium containingTyloxapol (10³ BCG-Tyl)

Group G: 10³ rBCG(mbtB) Tice—Grown in medium containing a highmycobactin J concentration (10⁶ rBCG(mbtB) Hi Fe)

Group H: 10³ rBCG(mbtB)30 II (pNBV1-30) Tice—Grown in medium containinga high mycobactin J concentration (10³ rBCG(mbtB)30 Hi Fe)

Group I: 10⁶ BCG Tice Parental Control—Grown in medium containingTyloxapol (10⁶ BCG-Tyl)

Group J: 10⁶ rBCG(mbtB) Tice—Grown in medium containing a highmycobactin J concentration (10⁶ rBCG(mbtB) Hi Fe)

Group K: 10⁶ rBCG(mbtB)30 II (pNBV1-30) Tice—Grown in medium containinga high mycobactin J concentration (10⁶ rBCG(mbtB)30 Hi Fe)

2. Cutaneous Delayed-type Hypersensitivity (DTH) to Purified RecombinantM. tuberculosis 30 kDa Major Secretory Protein (r30)

Ten weeks after immunization, 6 guinea pigs in each group were shavedover the back and injected intradermally with 10 μg of purifiedrecombinant M. tuberculosis 30 kDa major secretory protein (r30) in 100μl phosphate buffered saline. After 24 h, the diameter of erythema andinduration was measured. (A separate group of animals from the one usedin the challenge studies—see below—was used for skin-testing toeliminate the possibility that the skin-test itself might influence theoutcome). The results are summarized in Table 6.

TABLE 6 Cutaneous DTH - Experiment 5 Test Erythema Induration GroupStrain Antigen (mm ± SE) (mm ± SE) A Sham r30 2.1 ± 1.0 0 ± 0 B BCG r305.0 ± 1.3 0 ± 0 C rBCG30 r30 17.8 ± 2.1  16.5 ± 3.4  D rBCG30/hINFγ r304.3 ± 1.6 0 ± 0 E rBCG/hINFγ r30 6.6 ± 2.5 0 ± 0 F 10³ BCG-Tyl. r30 7.3± 1.6 1.7 ± 1.7 G 10³rBCG(mbtB) Hi Fe r30 1.5 ± 1.0 0 ± 0 H10³rBCG(mbtB)30 Hi Fe r30 8.8 ± 2.2 0 ± 0 I 10⁶BCG-Tyl. r30 4.0 ± 2.2 0± 0 J 10⁶rBCG(mbtB) Hi Fe r30 0 ± 0 0 ± 0 K 10⁶rBCG(mbtB)30 Hi Fe r3015.0 ± 0.8  10.3 ± 3.3 

These results showed that sham-immunized animals (Group A) and animalsimmunized with the parental BCG Tice strain (Groups B, F, and I) hadlittle or no induration upon testing with r30 whether the strain wasgrown in medium containing tyloxapol or not and whether or not a highdose was administered. Similarly, animals immunized with agrowth-restricted vaccine [rBCG(mbtB)] not overexpressing the 30 kDaprotein had no induration upon testing with r30, whether a low dose(Group G) or high dose (Group J) of the vaccine was administered. Incontrast, animals immunized with a recombinant BCG strain overexpressingr30 (Group C) had induration in response to r30. Similarly, animalsimmunized with the growth-restricted strain rBCG(mbtB)30, whenadministered as a high dose (Group K) showed induration upon testingwith r30. Animals immunized with a low dose of the growth-restrictedstrain rBCG(mbtB)30 (Group H) showed no induration upon testing withr30. Interestingly, as previously observed, the recombinant BCGexpressing both r30 and human interferon gamma (Group D) did not showinduration upon testing with r30, although it did display some erythema.

3. Protective Immunity to Aerosol Challenge

Ten weeks after immunization, the remaining animals in Groups A-K werechallenged with an aerosol generated from a 10 ml single-cell suspensioncontaining 7.5×10⁴ colony-forming units (CFU) of M. tuberculosis. (Priorto challenge, the challenge strain, M. tuberculosis Erdman strain [ATCC35801], had been passaged through outbred guinea pigs to maintainvirulence, cultured on 7H11 agar, subjected to gentle sonication toobtain a single cell suspension, and frozen at −70° C.). This aerosoldose delivered ˜10 live bacilli to the lungs of each animal. Theairborne route of infection was used because this is the natural routeof infection for pulmonary tuberculosis. A relatively large dose wasused so as to induce measurable clinical illness in 100% of controlanimals within a relatively short time frame (10 weeks). Afterwards,guinea pigs were individually housed in stainless steel cages containedwithin a laminar flow biohazard safety enclosure and allowed free accessto standard laboratory chow and water. The animals were observed forillness and weighed weekly for 10 weeks and then euthanized. The rightlung and spleen of each animal was removed and cultured for CFU of M.tuberculosis on Middlebrook 7H11 agar for two weeks at 37° C., 5%CO₂-95% air atmosphere. The results of the assay for CFU in the lungsand spleens are shown in Table 7.

TABLE 7 CFU in Lungs and Spleens - Experiment 3 Spleen Lung (Mean LogGroup Strain (Mean Log CFU ± SE) CFU ± SE) A Sham 6.63 ± 0.22 6.41 ±0.22 B BCG 5.17 ± 0.09 4.48 ± 0.06 C rBCG30 4.23 ± 0.13 3.47 ± 0.09 DrBCG30/hINFγ 4.03 ± 0.09 2.57 ± 0.26 E rBCG/hINFγ 5.11 ± 0.07 4.39 ±0.05 F 10³ BCG-Tyl. 4.96 ± 0.08 4.59 ± 0.07 G 10³rBCG(mbtB) Hi Fe 5.37 ±0.16 4.79 ± 0.18 H 10³rBCG(mbtB)30 Hi Fe 5.01 ± 0.16 4.63 ± 0.26 I10⁶BCG-Tyl. 5.17 ± 0.09 4.31 ± 0.08 J 10⁶rBCG(mbtB) Hi Fe 4.83 ± 0.124.23 ± 0.12 K 10⁶rBCG(mbtB)30 Hi Fe 4.56 ± 0.21 3.94 ± 0.21

These results showed that animals immunized with BCG or any recombinantBCG strain had much lower CFU in the lungs and spleens than the shamimmunized animals.

Animals immunized with the recombinant BCG strain secreting both the M.tuberculosis 30 kDa major secretory protein and human interferon gamma(rBCG30/hINFγ) had markedly fewer CFU in the lung and spleen thananimals immunized with rBCG30; animals immunized with rBCG30/hINFγ had0.2 logs fewer CFU in the lung and 0.9 logs fewer CFU in the spleen thanrBCG30-immunized animals.

Importantly, animals immunized with the recombinant BCG vaccinesecreting only human interferon gamma did not show protectionsignificantly different from BCG. Hence, it was the co-expression ofboth the 30 kDa protein and human interferon gamma that was necessaryfor the superior efficacy of the rBCG30/hINFγ vaccine.

Remarkably, animals immunized with the high dose of thegrowth-restricted recombinant BCG strain overexpressing the M.tuberculosis 30 kDa major secretory protein [rBCG(mbtB)30]) showed animpressive reduction in CFU in animal organs compared with BCG, whetheror not the BCG vaccine was grown in tyloxapol. There was no significantdifference in potency of BCG vaccines grown in the presence or theabsence of tyloxapol.

Experiment 6

1. Immunization of Animals

Specific-pathogen free 250-300 g outbred male Hartley strain guinea pigsfrom Charles River Breeding Laboratories, in groups of 15 or 21, weresham-immunized by intradermal administration of buffer (15 animalstotal) or immunized intradermally with 10³ or 10⁶ CFU of one of thefollowing strains (21 animals/group):

Group A: Sham-immunized (Sham)

Group B: 10³ BCG Tice Parental Control (10³ BCG)

Group C: 10³ rBCG30 Tice I (pSMT3-MTB30) (rBCG30)

Group D: 10⁶ BCG Tice Parental Control (10⁶ BCG)

Group E: 10⁶ rBCG(mbtB)30 II (pNBV1-30) Tice—Grown in medium containinga high mycobactin concentration (10⁶ rBCG(mbtB)30 Hi Fe)

Group F: 10³ rBCG(panCD)30 Tice(10³ rBCG(panCD)30)

Group G: 10⁶ rBCG(panCD)30 Tice (10⁶ rBCG(panCD)30)

Group H: 10³ rBCG(panCD)30 Tice—Animals fed diet high in pantothenate(10³ rBCG(panCD)30+Diet)

Group I: 10⁶ rBCG(panCD)30 Tice—Animals fed diet high in pantothenate(10⁶ rBCG(panCD)30+Diet)

Group J: 10⁶ rBCG(trpD)30 (pSMT3-MTB30) Tice (10⁶ rBCG(trpD)30)

Group K: 10⁸ rBCG(panCD)30 Tice (10⁸ rBCG(panCD)30)

2. Cutaneous Delayed-type Hypersensitivity (DTH) to Purified RecombinantM. tuberculosis 30 kDa Major Secretory Protein (r30)

Ten weeks after immunization, 6 guinea pigs in each group were shavedover the back and injected intradermally with 10 μg of purifiedrecombinant M. tuberculosis 30 kDa major secretory protein (r30) in 100μl phosphate buffered saline. After 24 h, the diameter of erythema andinduration was measured. (A separate group of animals from the one usedin the challenge studies—see below—was used for skin-testing toeliminate the possibility that the skin-test itself might influence theoutcome). The results are summarized in Table 8.

TABLE 8 Cutaneous DTH - Experiment 6 Test Erythema Induration GroupStrain Antigen (mm ± SE) (mm ± SE) A Sham r30  1.5 ± 1.0 0 ± 0 B 10³ BCGr30  8.5 ± 1.8 0 ± 0 C rBCG30 r30 10.7 ± 2.3 2.5 ± 2.5 D 10⁶ BCG r3011.0 ± 0.8 0 ± 0 E 10⁶ rBCG(mbtB)30 r30 14.0 ± 0.9 10.0 ± 3.2  Hi Fe F10³ rBCG(panCD)30 r30  2.5 ± 1.7 0 ± 0 G 10⁶ rBCG(panCD)30 r30 12.8 ±0.8 2.3 ± 2.3 H 10³ rBCG(panCD)30 + r30  1.3 ± 1.3 0 ± 0 Diet I 10⁶rBCG(panCD)30 + r30 11.0 ± 1.6 5.0 ± 3.2 Diet J 10⁶ rBCG(trpD)30 r3012.0 ± 2.7 5.8 ± 3.7 K 10⁸ rBCG(panCD)30 r30 11.7 ± 1.0 6.3 ± 2.9

These results showed that sham-immunized animals (Group A) and animalsimmunized with the parental BCG Tice strain (Groups B and D) had noinduration upon testing with r30 whether the dose was high or low.Similarly, animals immunized with a low dose of rBCG(panCD)30 with orwithout a dietary supplement had no induration upon testing with r30. Incontrast, animals immunized with rBCG30 as well as with high doses (10⁶or 10⁸) of rBCG(mbtB)30 Hi Fe, rBCG(panCD)30, and rBCG(trpD)30 hadrelatively high amounts of induration in response to r30.

3. Protective Immunity to Aerosol Challenge.

Ten weeks after immunization, the remaining animals in Groups A-K werechallenged with an aerosol generated from a 10 ml single-cell suspensioncontaining 7.5×10⁴ colony-forming units (CFU) of M. tuberculosis. (Priorto challenge, the challenge strain, M. tuberculosis Erdman strain [ATCC35801], had been passaged through outbred guinea pigs to maintainvirulence, cultured on 7H11 agar, subjected to gentle sonication toobtain a single cell suspension, and frozen at −70° C.). This aerosoldose delivered ˜10 live bacilli to the lungs of each animal. Afterwards,guinea pigs were individually housed in stainless steel cages containedwithin a laminar flow biohazard safety enclosure and allowed free accessto standard laboratory chow and water. The animals were observed forillness and weighed weekly for 10 weeks and then euthanized. The rightlung and spleen of each animal was removed and cultured for CFU of M.tuberculosis on Middlebrook 7H11 agar for two weeks at 37° C., 5%CO₂-95% air atmosphere. The results of the assay for CFU in the lungsand spleens are shown in Table 9.

TABLE 9 CFU in Lungs and Spleens - Experiment 4 Lung Spleen (Mean Log(Mean Log Group Strain CFU ± SE) CFU ± SE) A Sham 6.73 ± 0.16 6.74 ±0.15 B 10³ BCG 4.22 ± 0.08 3.95 ± 0.08 C rBCG30 3.48 ± 0.11 2.34 ± 0.14D 10⁶ BCG 4.09 ± 0.07 3.80 ± 0.07 E 10⁶ rBCG(mbtB)30 Hi Fe 4.24 ± 0.053.78 ± 0.05 F 10³ rBCG(panCD)30 5.40 ± 0.06 5.28 ± 0.06 G 10⁶rBCG(panCD)30 4.72 ± 0.10 4.77 ± 0.13 H 10³ rBCG(panCD)30 + Diet 5.07 ±0.08 4.94 ± 0.11 I 10⁶ rBCG(panCD)30 + Diet 4.57 ± 0.10 4.46 ± 0.09 J10⁶ rBCG(trpD)30 4.83 ± 0.09 4.67 ± 0.11 K 10⁸ rBCG(panCD)30 4.27 ± 0.074.01 ± 0.10

These results showed that animals immunized with BCG or any recombinantBCG strain had much lower CFU in the lungs and spleens than the shamimmunized animals.

Animals immunized with the growth-restricted strain rBCG(mbtB)30 Hi Fehad CFU comparable to BCG-immunized animals in their organs.

Animals immunized with 10³, 10⁶, and 10⁸ rBCG(panCD)30 showed adose-dependent effect on CFU in their organs; the higher the dose thelower the CFU counts. The effect on dose was statistically significantin both the lung and spleen (P<0.0001 by ANOVA).

Animals immunized with rBCG(panCD)30 and fed a diet rich in pantothenatehad fewer CFU than animals immunized with the same dose of rBCG(panCD)30and not fed a diet rich in pantothenate. The effect on diet wasstatistically significant (P=0.008 in the lung and P=0.003 in the spleenby ANOVA).

Example 5 Clearance of Vaccines in Guinea Pigs

Specific-pathogen free 250-300 g outbred male Hartley strain guinea pigsfrom Charles River Breeding Laboratories, in groups of 24, wereimmunized intradermally with 10⁶ CFU of one of the following strains:

Group A: 10⁶ BCG Tice Parental Control (BCG)

Group B: 10⁶ rBCG(mbtB)30 II (pNBV1-30) Tice—Grown in medium containinga high mycobactin J concentration (rBCG(mbtB)30)

Group C: 10⁶ rBCG(panCD)30 Tice (rBCG(panCD)30)

At 1, 2, 3, 4, 6, 8, 10, and 15 weeks after immunization, three animalsper group were euthanized and CFU of BCG, rBCG(mbtB)30, andrBCG(panCD)30 in the lung, spleen, and lymph nodes were assayed (FIGS. 9and 10; the results for BCG-immunized animals are repeated in eachfigure). Data are presented as the mean Log CFU±SE. The limit ofdetection was 1 Log CFU.

These results showed that the persistence of the growth-restrictedrBCG(mbtB)30 strain was much less than the persistence of BCG in guineapig organs (FIG. 9).

The rBCG(panCD)30 strain also showed much less persistence than BCG inguinea pig organs (FIG. 10).

Example 5 Virulence of Vaccines in SCID Mice

Fox Chase SCID mice (CB-17/lcr-Prkdc^(scid)/Crl) from Charles RiverBreeding Laboratories in groups of 20 were challenged by intravenoustail vein injection with 10⁶ CFU or 10⁸ CFU of one of the followingstrains or injected with buffer (PBS):

Group A: Sham-challenged with PBS buffer (Sham)

Group B: 10⁶ BCG Tice Parental Control (10⁶ BCG)

Group C: 10⁶ rBCG(mbtB)30 II (pNBV1-30) Tice—Grown in medium containinga high mycobactin concentration (10⁶ rBCG(mbtB)30)

Group D: 10⁶ rBCG(panCD)30 Tice (10⁶ rBCG(panCD)30)

Group E: 10⁸ rBCG(mbtB)30 II (pNBV1-30) Tice—Grown in medium containinga high mycobactin concentration (10⁸ rBCG(mbtB)30)

Group F: 10⁸ rBCG(panCD)30 Tice (10⁸ rBCG(panCD)30)

Group G: 10⁸ BCG Tice Parental Control (10⁸ BCG)

Survival was monitored for 25 weeks (FIGS. 11 and 12; the results forsham and BCG-immunized animals are repeated in each figure).

These results showed that the growth-restricted rBCG(mbtB)30 strain wasmuch safer than BCG in the immunocompromised SCID mouse (FIG. 11). At adose of 10⁶ CFU, the vaccine was essentially avirulent, whereas BCG atthis dose killed all animals by 22 weeks. At an extremely high dose of10⁸ CFU, the vaccine was somewhat more virulent than sham immunization,but still much less virulent than BCG, which killed all animals by 15weeks.

The rBCG(panCD)30 strain was also much safer than BCG, and wasessentially avirulent at challenge doses of both 10⁶ CFU and 10⁸ CFU(FIG. 12).

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe following specification and attached claims are approximations thatmay vary depending upon the desired properties sought to be obtained bythe present invention. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of the invention areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical value, however,inherently contains certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

The terms “a” and “an” and “the” and similar referents used in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein is merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is hereindeemed to contain the group as modified thus fulfilling the writtendescription of all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

1. An immunogenic composition comprising: a growth regulatablerecombinant Bacille Calmette Guérin (rBCG) expressing at least oneMycobacteria major extracellular protein selected from the groupconsisting of 12 kDa protein, 14 kDa protein, 16 kDa protein, 23.5 kDaprotein, 24 kDa protein, 30 kDa protein, 32A kDa protein, 32B kDaprotein, 45 kDa protein, 58 kDa protein, 71 kDa protein, 80 kDa protein,and 110 KD protein, and combinations thereof, and wherein saidMycobacteria major extracellular proteins are over-expressed andsecreted.
 2. The immunogenic composition according to claim 1 wherein atleast one of said at least one Mycobacteria major extracellular proteinsare expressed on one or more extrachromosomal nucleic acid sequences. 3.(canceled)
 4. The immunogenic composition according to claim 2 whereineach of said at least one Mycobacteria major extracellular proteins areexpressed from different extrachromosomal nucleic acid sequences.
 5. Theimmunogenic composition according to claim 1 wherein at least one ofsaid at least one Mycobacteria major extracellular proteins areintegrated into the rBCG genome under the control of a strong promoterand over-expressed.
 6. The immunogenic composition according to claim 5wherein more than one of said at least one Mycobacteria majorextracellular proteins are integrated into the rBCG genome under thecontrol of a strong promoter and over-expressed.
 7. The immunogeniccomposition according to claim 1 wherein said major extracellularproteins are non-fusion proteins.
 8. (canceled)
 9. The immunogeniccomposition according to claim 1 wherein said growth regulatable rBCG isselected from the group consisting of auxotrophs and metabolicallyimpaired mutants.
 10. The immunogenic composition according to claim 9wherein said metabolically impaired mutant is a siderophore mutant. 11.The immunogenic composition according to claim 10 wherein saidsiderophore is a mycobactin or an exochelin.
 12. The immunogeniccomposition according to claim 9 wherein said growth regulatable rBCG isan auxotroph and wherein pantothenic acid is used to regulate growth ofsaid auxotroph.
 13. The immunogenic composition according to claim 1wherein said Mycobacteria major extracellular protein is from aMycobacteria species selected from the group consisting of Mycobacteriumtuberculosis, Mycobacterium bovis, Mycobacterium leprae, andMycobacterium avium intracellulare.
 14. (canceled)
 15. The immunogeniccomposition according to claim 1 wherein said Mycobacteria majorextracellular protein is the 30 kDa protein.
 16. The immunogeniccomposition according to claim 1 further expressing at least onecytokine selected from the group consisting of interferon gamma,interleukin-2, interleukin-12, interleukin-4 receptor and granulocytemacrophage colony stimulating factor, and combinations thereof. 17.(canceled)
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)22. (canceled)
 23. (canceled)
 24. An immunogenic composition comprisinga growth regulatable recombinant attenuated intracellular pathogenwherein said growth regulatable recombinant attenuated intracellularpathogen expresses at least one major extracellular protein of anintracellular pathogen wherein a nucleic acid sequence encoding for saidat least one major extracellular protein is incorporated into theintracellular pathogen's chromosome(s) under a strong promoter such thatthe major extracellular protein is over-expressed.
 25. The immunogeniccomposition of claim 24 wherein said growth regulatable recombinantattenuated intracellular pathogen is of the same species as theintracellular pathogen against which the immunogenic composition isdirected.
 26. The immunogenic composition of claim 24 wherein saidgrowth regulatable recombinant attenuated intracellular pathogen is of adifferent species than the intracellular pathogen against which theimmunogenic composition is directed.
 27. The immunogenic composition ofclaim 24 wherein said growth regulatable recombinant attenuatedintracellular pathogen is selected from the group consisting ofMycobacterium bovis, M. tuberculosis, M. leprae, M. kansasii, M. avium,Mycobacterium sp., Legionella pneumophila, L. longbeachae, L. bozemanii,Legionella sp., Rickettsia rickettsii, Rickettsia typhi, Rickettsia sp.,Ehrlichia chaffeensis, Ehrlichia phagocytophila geno group, Ehrlichiasp., Coxiella burnetii, Leishmania sp, Toxpolasma Trypanosoma cruzi,Chlamydia pneumoniae, Chlamydia sp, Listeria monocytogenes, Listeria sp,Histoplasma sp., Francisella tularensis, Brucella species, Yersiniapestis, Bacillus anthracis, and Salmonella typhi.
 28. The immunogeniccomposition of claim 24 wherein said at least one major extracellularprotein is from an intracellular pathogen selected from the groupconsisting of Mycobacterium bovis, M. tuberculosis, M. leprae, M.kansasii, M. avium, Mycobacterium sp., Legionella pneumophila, L.longbeachae, L. bozemanii, Legionella sp., Rickettsia rickettsii,Rickettsia typhi, Rickettsia sp., Ehrlichia chaffeensis, Ehrlichiaphagocytophila geno group, Ehrlichia sp., Coxiella burnetii, Leishmaniasp., Toxpolasma gondii, Trypanosoma cruzi, Chlamydia pneumoniae,Chlamydia s.p, Listeria monocytogenes, Listeria sp., Histoplasma sp.,Francisella tularensis, Brucella species, Yersinia pestis, Bacillusanthracis, and Salmonella typhi.
 29. (canceled)
 30. (canceled)
 31. Animmunogenic composition comprising a growth regulatablesiderophore-dependent rBCG wherein said growth regulatable rBCGexpresses the 30 kDa Mycobacteria major extracellular protein and anucleic acid sequence encoding for said 30 kDa Mycobacteria majorextracellular protein is incorporated into the intracellular pathogen'schromosome(s) under a strong promoter such that said 30 kDa Mycobacteriamajor extracellular protein is over-expressed and wherein mycobactin isused to allow growth of the rBCG in vitro.
 32. The immunogeniccomposition of claim 31 wherein said growth regulatablesiderophore-dependent rBCG further expresses interferon gamma.